1. Field of the Invention
The present invention relates to the identification of the sequence of the pS2 protein secreted in different human tissues and humors, and to peptide fragments of this protein; the present invention further relates to polyclonal and monoclonal antibodies directed against pS2 and against peptide fragments of the pS2 protein, and to the applications of this protein and these peptides and antibodies to the diagnosis and detection of various pathological conditions and especially hormone-dependent breast cancers and stomach cancers or ulcers.
2. Discussion of the Background
The fact that breast cancer is hormone-dependent has been known since 1896, when Beatson [Lancet (1986), 2, 104-107] reported two observations of the regression of inoperable tumors after ovariectomy in women who were still having menstrual periods. It is now well established that about one third of breast cancers respond to hormones and regress following a variety of hormonal manipulations.
Until recent years, there was no biochemical test for identifying women who were suffering from hormone-dependent cancer and who could therefore benefit from endocrinotherapy. In 1971, Jensen et al. [NATL. CANCER INSTI. MONOGR. (1971) 34, 55-70] were the first to show that measurement of the estrogen receptors in a tumor specimen could be useful for predicting the response to adrenalectomy. This observation has since been amply confirmed as 50 to 65% of women whose tumors contain estrogen receptors (ER's) respond to endocrinotherapy, whereas those whose tumors do not contain estrogen receptors have only a 10% chance, at best, of being helped by hormone treatment.
In fact, if cancerous cells possess sites having a strong affinity for a hormone (i.e. a receptor), such sites normally being present in the mammary gland, their growth, like that of normal cells, can be regulated by the hormonal environment. Conversely, if cancerous cells lose their receptors during the malignant transformation, they are no longer recognized as target cells. However, the result of determination of the estrogen receptors (ER's) does not permit a perfect prediction of the response to hormonotherapy as only 55% to 65% of women whose tumors possess receptors respond favorably to hormone treatment. One of the explanations lies in the fact that, in the course of dedifferentiation, some tumors may lose their estrogen receptors. If these receptors persist, the tumors may retain the capacity to bind estradiol, but are incapable of carrying through the subsequent steps of the estrogenic action. Both cases involve tumors which are hormonally autonomous or hormone-resistant. This last hypothesis was proved by going beyond the initial step, i.e. binding to the cytosol receptor, and investigating the end products of the intracellular action of estrogens. This is the case, for example, of the progesterone receptor (PR) as its synthesis is dependent on estrogens in MCF-7 cells (cell line derived from a human breast cancer) and probably in human breast cancer cells in vivo. In actual fact, if the proportion of progesterone receptors is taken into account, the remission levels of cancers under hormone treatment are of the order of 65%. Numerous clinical trials have established that 80% of women whose tumors possess both receptors respond to hormonotherapy. By contrast, if the tumor contains estrogen receptors but not progesterone receptors, the probability of response is no more than one third of cases. The progesterone receptors (PR's) therefore provide additional information for prediction of the therapeutic response.
Estrogens stimulate the synthesis of a large number of proteins which are released in the incubation medium of cell lines such as MCF-7. Most of the proteins secreted are detected in the incubation medium with or without estradiol, but their activity is very considerably increased if this hormone is present. They correspond to molecular weights of 37,000, 46,000, 54,000 and 60,000M [MAIRESSE et al. in RECENT RESULTS IN CANCER RESEARCH, G. LECLERCQ, S. TOMA, R. PARIDAENS, J. C. HEUSEN, vol. 91, (1984) 301-306]. Some of them (46,000, 54,000 and 60,000M) are identical to the cytosol proteins. A protein of 50,000M is more abundant in the incubation medium treated with estradiol, but MAIRESSE's work demonstrates that this involves stimulation of the secretion of this protein under the action of a hormone, rather than induction. Furthermore, these stimulated proteins also exist in the incubation medium of MCF-7 cells and are also present in the incubation medium of estrogen-independent Evsa-T cells, but the estradiol in this last medium has no effect on the syntheses and/or their secretion. This is not therefore a case of the induction of a new product under hormonal influence, but only an increase in the concentration of an existing product.
ROCHEFORT (same publication as above, p. 289-294) detected a high level of 52K protein in the incubation medium and showed that the induction of this protein was specific for the action of estradiol at physiological concentrations, whereas progesterone and dexamethasone were inactive. Tamoxifen, which inhibits cell growth, does not induce secretion of the 52K protein and prevents the action of estradiol in a molar ratio of 10. One of its metabolites, monohydroxytamoxifen, is 200 times more active than tamoxifen in blocking cell growth and the secretion of 52K in MCF-7 cells. More recently, this same team found that in a variant of MCF-7 cells, namely R-27 cells, which possess ER's and PR's but whose growth escapes the action of estrogen inhibitors, the 52K protein continues to be secreted in the presence of tamoxifen or monohydroxytamoxifen.
A team of researchers, including some of the inventors of the present patent application, set out to achieve the expression of specific genes. Starting with a cDNA library built up from MCF-7 cells induced by estradiol, it was possible to carry out differential cloning of the cDNA's corresponding to the mRNA synthesized in the presence of this hormone. With the aid of a cDNA probe produced from cells growing in the presence and absence of hormone, a cDNA clone corresponding to an mRNA which is present only in MCF-7 cells cultivated in the presence of estradiol could be isolated. It was called pS2. The authors deduced, from the determination of the nucleotide sequence of the cloned cDNA, that it is a protein comprising 84 amino acids and having a low molecular weight of 9140 Daltons [JAKOWLEW et al., NUCLEIC ACIDS RES. (1984) 12, 2861-2878]. The hormonal regulation exerted on the pS2 gene is situated at the transcriptional level. The gene is not transcribed in the absence of estradiol, whereas there is a distinct accumulation of mRNA eight hours after the hormone has been added to the culture medium. However, the authors had not yet isolated the pS2 protein. Estradiol-induced screening of the cDNA library built up from MCF-7 cells made it possible also to isolate two other clones, 36 B4 and 3 A5. No hormonal regulation is exerted at the transcriptional level of their corresponding gene. These two clones were called "constant" clones. The 36 B4 and 3 A5 probes can therefore be used to assess the amount of total mRNA's present, whereas the pS2 probe corresponds to a specific estrogen-induced RNA of MCF-7 cells. pS2 RNA is not present in the RNA extracted from T 47 D human breast cancer cells, which contain both estrogen and progesterone receptors but in which the presence of the latter receptor is constitutive. Conversely, 36 B4 RNA is present in T 47 D cells.
JELTSCH et al. [NUCLEIC ACID RES. (1987), 15, p. 1401-1414] subsequently cloned the human pS2 gene from DNA of placental cells and cells of the MCF-7 line, studied its structure and established and hence verified the nucleotide sequence of the pS2 gene on the basis of the pS2M clone obtained from the said cell line, and on the basis of the pS2P clone obtained from placental cells.
The observation that pS2 RNA is expressed in the MCF-7 cell line derived from a human breast cancer, but is not expressed in the T 47D cell line, indicates a method of identifying hormone-dependent breast cancers. It is for this reason that the inventors attempted to check whether the expression of the pS2 gene can constitute an additional marker for the detection of hormone-dependent breast cancers.
More recently [RIO et al., SCIENCE (1988) 241, p. 705-707], the pS2 protein was detected in the mucosa cells of the gastric epithelium. The protein secreted in the gastric fluid has an identical electrophoretic migration to that observed for the protein secreted by MCF-7 cells, and the work reported in the article cited above shows that the size and sequence of the mRNA' s isolated from the two tissues are strictly identical.
It should be recalled that sequence (I), determined for the pS2 protein from mRNA, comprises 84 amine acids, has a molecular weight of the order of 9140 Daltons and is as follows:
__________________________________________________________________________ 1 10 MET ALA THR MET GLU ASN LYS VAL ILE CYS ALA LEU VAL LEU 20 VAL SER MET LEU ALA LEU GLY THR LEU ALA GLU ALA GLN THR 30 40 GLU THR CYS THR VAL ALA PRO ARG GLU ARG GLN ASN CYS GLY 50 PHE PRO GLY VAL THR PRO SER GLN CYS ALA ASN LYS GLY CYS 60 70 CYS PHE ASP ASP THR VAL ARG GLY VAL PRO TRP CYS PHE TYR 80 PRO ASN THR ILE ASP VAL PRO PRO GLU GLU GLU CYS GLU PHE (I) __________________________________________________________________________
Now, the inventors have been able to establish that this is not the secreted form of the pS2 peptide.